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> On the subject of surface vs DNA analysis: unfortunately I have no
> good suggestions to add but would like to comment on a couple of
> suggestions made so far. The problem of PI (DNA) vs PE (surface) is
> that the intense PI fluorescence leaks into the PE detector. Nothing
> you do to the PI detector optical filters will change that; going to
> a longer wavelength PI filter doesn't change the PI emission spectrum.
> The PE detector will be subjected to just the same amount of
> leakage. On the other hand, going to a *shorter* wavelength *PE
> detector* optical filter may indeed take you into an area of better
> PE-to-PI detection ratio.
I agree with this, but would like to add a couple of points from our
own experience. We switched to using 7-amino actinomysin-D (7AAD)
instead of PI, because we found less problems with compensation, mostly I
think because it was easier to control a weaker signal, although it
also has the advantage of not needing RNAse treatment. Again we used
a long band-pass filter to prevent PE into 7AAD being a problem.
7AAD into PE was still not perfect but we were comparing a range of
activation associated markers on T cells such as CD69 CD25, CD71 etc
with CD4 and CD8 labelling. We got around the problem by always
using FITC for the dodgy (activation) markers, and PE for CD4 or CD8.
This produced entirely acceptable results for all three fluorescence
channels. If you have two dodgy surface markers.......
well I wish you luck!!!
Have fun
Mike
which of course give a much better seperation.
...........................................................
Mike Salmon
Department of Rheumatology
The Medical School
University of Birmingham
Birmingham B15 2TT
United Kingdom
Tel: 44 (0)121 414 6780
Fax: 44 (0)121 414 6794
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